Our laboratory has conducted two research programs at the interface of chemistry and evolution. In the first program, we developed new approaches to protein evolution and protein delivery that have dramatically increased their effectiveness. In the second program, we developed a new approach to the synthesis and discovery of bioactive small molecules that combines powerful aspects of biological evolution with synthetic organic chemistry. The resulting method of DNA-templated synthesis has enabled DNA sequences encoding synthetic molecules to undergo translation, selection, and amplification paralleling biological evolution. This proposal seeks to integrate these two research programs into a single effort under a Maximizing Investigators' Research Award (MIRA). In the first program, we developed a system that enables proteins to evolve continuously in the laboratory, requiring virtually no researcher intervention. The resulting system, phage-assisted continuous evolution (PACE), allows proteins to undergo directed evolution at a rate ~100-fold faster than conventional methods. We propose to apply these developments to continuously evolve four classes of proteins or RNAs, each with the ability to manipulate the covalent structure of genes or gene products, and each with potential relevance to the development of next-generation human therapeutics: recombinase enzymes that insert DNA of interest into safe-harbor loci in the human genome, proteases that specifically cleave disease-associated proteins, orthogonal Cas9 (CRISPR) nucleases with altered PAM specificities and enhanced activities, and smart Cas9 guide RNAs that mediate genome engineering only in those cells that are in specific disease-associated cell states. Success would establish the novel therapeutic potential of these proteins and RNAs to address a wide range of human diseases, including many human genetic disorders. In the second program, we developed the foundations of DNA-templated synthesis, generated libraries of DNA-templated small molecules, and performed in vitro selections on these libraries for affinity to an initial set of targets of biomedical interest. The results led t the discovery of novel kinase and protease inhibitors with remarkable selectivity and potency, including the first physiological inhibitor of insulin-degrading enzyme (IDE). We used this compound in mice to validate inhibition of insulin degradation as a potential therapeutic strategy for improving glucose tolerance. We propose to develop second-generation IDE inhibitors with increased therapeutic potential, to explore our recent discovery that IDE inhibition can lower blood pressure in vivo, and to expand the application of this approach by creating a new DNA-templated library of >250,000 macrocycles and to selecting this library for binding to >150 protein targets implicated in human disease. These efforts will collectively result in the evaluation of more than 30,000,000 potential small molecule-protein interactions in a manner that leverages the remarkable efficiency of in vitro selection, PCR, and modern DNA sequencing.